Wet Lab Experiments

Our 2 main wet lab projects were the transformation of Bacillus Subtilis and assembly of our plasmids using Gibson Assembly. We first conducted growth curve assays of our two strains (3A37 and 1A436) of Bacillus Subtilis.

== Electroporation Protocol

==

Purpose Electroporation promises better transformation efficiency than starvation protocols and takes less time and is easier to execute. This protocol outlines how to use the Life Technologies Cell-Porator machine found in Bindley 222.

Solutions Growth medium: LB medium containing 0.5 M sorbitol Washing solution: 0.5 M sorbitol, 0.5 M mannitol, 10 % glycerol Electroporation solution: 0.5 M sorbitol, 0.5 M mannitol, 10 % glycerol

Outgrowth medium: LB medium containing 	0.5 M sorbitol and 0.38 M mannitol

Materials • Life Technologies Cell-Porator • Cell-Porator chambers (4) • Competent cells • Micropipette and tips • SOC solution

Methods Making electrocompetent cells: 1. Dilute an overnight culture of Bacillus subtilis 16-fold in growth medium and grow at 37 °C to an O.D.600 of 0.85-0.95. 2. Cool the cells on ice-water for 10 min. and harvest by centrifugation at 4 °C and 5000 x g for 5 min. 3. Wash cells four times in ice-cold electroporation medium. 4. Suspend the cells in 1/40 of the culture volume of the electroporation solution with a cell concentration of 1-1.3 x 1010 cfu/ml. 5. The competent cells can be stored at –80 °C until use with some decrease in transformation efficiency.

Electroporation of cells: 1. Add 1 µl (50 ng/µl) plasmid DNA to 60 µl of electrocompetent cells. Homogenize by gently mixing with pipette several times. Transfer mixture into a prechilled cuvette. Incubate for 1-1.5 min. 2. Place ice water in chamber under cell holder rack 3. Place electroporator chambers on ice 4. Turn on voltage booster and controller using switches on back 5. Set to these conditions: 6. Voltage Booster: Level 4 7. Charge rate: fast 8. Volts: Low 9. Capacitance: 330 10. Pipette 20μL of competent cells with DNA between electrodes 11. Fill all 4 slots in chamber 12. Close chamber, plug in power source 13. Make sure chamber selector is on the correct chamber 14. Press up on the controller until voltage reaches 405 15. At 405, switch from charge to arm and press trigger for 1 second 16. Release trigger and switch arm to charge *If there is too much salt in the DNA, a pop will happen and the liquid will not be suspended between the electrodes 17. Immediately add 1 ml outgrowth medium and incubate for 3 h at 37 °C. 18. Plate onto selective LB agar plates and incubate overnight at 37 °C.

== Phytosiderrophore Production Assay ==

O-CAS Siderophore Assay

Introduction The CAS assay includes inhibitory compounds that negatively impact the growth of gram-positive bacteria (e.g. Bacillus subtilis) and fungi. Therefore, the O-CAS assay must be used.

Materials • 20% glycerol solution • Chrome azurol S (CAS) • HDTMA • PIPES • Agarose

Procedure 1. Plates containing growth medium were stored at 28°C for 24h 2. Bacteria were preserved in 20% glycerol solution at -20°C 3. The CAS medium is prepared as follows: a. Chrome azurol S (CAS) 60.5 mg b. hexadecyltrimetyl ammonium bromide (HDTMA) 72.9 mg c. Piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) 30.24 g d. 1 mM FeCl3• 6H2O in 10 mM HCl 10 mL. e. Agarose (0.9%, w/v) was used as gelling agent 4. Apply 10mL of medium over standard Petri dish (or 30mL over large Petri dish) 5. Color will change after 15 min if siderophores are detected

Paper http://ac.els-cdn.com/S0167701207001315/1-s2.0-S0167701207001315-main.pdf?_tid=0f6ae27a-f585-11e3-a635-00000aacb361&acdnat=1402943939_6d4bbe673ecc5dfa11e7309507a2c4e8

We synthesized our 2 plasmids using Integrated DNA Technologies (IDT). We currently have them as gBlocks and are trouble shooting the Gibson Assembly.